A neoplasm is an abnormal mass of tissue resulting from the abnormal proliferation of cells. The growth of a neoplasm exceeds and is uncoordinated with that of the normal (i.e., non-neoplastic) tissues around it. Neoplasms typically cause a lump or a tumor and may be benign, pre-malignant, or malignant. The initial growth of a neoplasm is dependent upon adequate supply of growth factors and the removal of toxic molecules. The expansion of tumor mass beyond 2 mm in diameter depends on the development of angiogenesis to produce adequate blood supply. The induction of angiogenesis is mediated by multiple molecules that are released by both tumor cells and host cells, including endothelial cells, epithelial cells, mesothelial cells, and leukocytes. Angiogenesis comprises sequential processes emanating from microvascular endothelial cells. As it expands, the tumor (primary or secondary) can also cause certain symptoms, such as discomfort (e.g., the feeling of a lump), pain and bleeding. After angiogenesis begins, tumor cell invasion of the tissue surrounding the primary tumor and penetration of blood and lymph vessels is central to the whole phenomenon of metastasis.
Once tumor cells detach from the primary tumor, they must invade the host stroma to penetrate lymphatics and blood vessels. To do so, tumor cells must penetrate basement membranes surrounding blood vessels. Basement membranes and connective tissue extracellular matrix (ECM) is comprised of four major groups of molecules: collagens, elastins, glycoproteins, and proteoglycans. The degradation of the ECM and basement membrane components by tumor cells is an important prerequisite for invasion and metastasis.
Cancer metastasis is comprised of multiple complex, interacting, and interdependent steps, each of which is rate-limiting, since a failure to complete any of the steps prevents the tumor cell from producing a metastasis. The tumor cells that eventually give rise to metastases must survive a series of potentially lethal interactions with host homeostatic mechanisms. The balance of these interactions can vary among different patients with different neoplasms or even among different patients with the same type of neoplasm.
The important steps in the formation of a metastasis are similar in all tumors and comprises the following:
1. After neoplastic transformation, progressive proliferation of neoplastic cells is initially supported with nutrients supplied from the organ microenvironment by diffusion.
2. Neovascularization or angiogenesis must take place for a tumor mass to exceed 1 or 2 mm in diameter. The synthesis and secretion of different angiogenic molecules and suppression of inhibitory molecules are responsible for the establishment of a capillary network from the surrounding host tissue.
3. Some tumor cells can down regulate expression of cohesive molecules and have increased motility, thus can detach from the primary lesion. Invasion of the host stroma by some tumor cells occurs by several parallel mechanisms. Capillaries and thin-walled venules, like lymphatic channels, offer very little resistance to penetration by tumor cells and provide the most common pathways for tumor cell entry into the circulation.
4. Detachment and embolization of single tumor cells or cell aggregates occur next, the vast majority of circulating tumor cells being rapidly destroyed.
5. Once the tumor cells have survived circulation, they must arrest in the capillary beds of distant organs by adhering either to capillary endothelial cells or to exposed subendothelial basement membranes.
6. Tumor cells (especially those in aggregates) can proliferate within the lumen of the blood vessel, but the majority move into the organ parenchyma by mechanisms similar to those operative during invasion.
7. Tumor cells bearing appropriate cell surface receptors respond to paracrine growth factors and hence proliferate in the organ parenchyma.
8. The metastatic cells must evade destruction by host defenses that include specific and nonspecific immune responses.
9. To exceed a mass of 1 to 2 mm in diameter, metastasis must develop a vascular network.
There are several chemotherapy drugs and anti-cancer therapies currently used to treat a variety of cancers by, for example, damaging DNA in the cancer cell to preventing the cell from reproducing. Chemotherapy drugs can be divided into several groups based on factors such as how they work, their chemical structure, and their relationship to another drug. Because some drugs act in more than one way, they may belong to more than one group.
Alkylating agents directly damage DNA to prevent the cancer cell from reproducing. As a class of drugs, these agents are not phase-specific; in other words, they work in all phases of the cell cycle. Alkylating agents are used to treat many different cancers, including acute and chronic leukemia, lymphoma, Hodgkin disease, multiple myeloma, sarcoma, as well as cancers of the lung, breast, and ovary. Because these drugs damage DNA, they can cause long-term damage to the bone marrow. In a few rare cases, this can eventually lead to acute leukemia. The risk of leukemia from alkylating agents is “dose-dependent,” meaning that the risk is small with lower doses, but goes up as the total amount of drug used gets higher. The risk of leukemia after alkylating agents is highest 5 to 10 years after treatment. There are many different alkylating agents, including: nitrogen mustards, such as mechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide (Cytoxan®), ifosfamide, and melphalan; nitrosoureas, such as streptozocin, carmustine (BCNU), and lomustine; alkyl sulfonates, which include busulfan; triazines, such as dacarbazine (DTIC), and temozolomide (Temodar®); and ethylenimines such as thiotepa and altretamine (hexamethylmelamine). The platinum drugs (cisplatin, carboplatin, and oxalaplatin) are sometimes grouped with alkylating agents because they kill cells in a similar way. These drugs are less likely than the alkylating agents to cause leukemia.
Antimetabolites are a class of drugs that interfere with DNA and RNA growth by substituting for the normal building blocks of RNA and DNA. These agents damage cells during the S phase. They are commonly used to treat leukemias, tumors of the breast, ovary, and the intestinal tract, as well as other cancers. Examples of antimetabolites include 5-fluorouracil (5-FU), capecitabine (Xeloda®), 6-mercaptopurine (6-MP), methotrexate, gemcitabine (Gemzar®), cytarabine (Ara-C®), fludarabine, and pemetrexed (Alimta®).
Anthracyclines are anti-tumor antibiotics that interfere with enzymes involved in DNA replication. These agents work in all phases of the cell cycle. Thus, they are widely used for a variety of cancers. A major consideration when giving these drugs is that they can permanently damage the heart if given in high doses. For this reason, lifetime dose limits are often placed on these drugs. Examples of anthracyclines include daunorubicin, doxorubicin (Adriamycin®), epirubicin, and idarubicin. Other anti-tumor antibiotics include the drugs actinomycin-D, bleomycin, and mitomycin-C.
Mitoxantrone is an anti-tumor antibiotic that is similar to doxorubicin in many ways, including the potential for damaging the heart. This drug also acts as a topoisomerase II inhibitor, and can lead to treatment-related leukemia. Mitoxantrone is used to treat prostate cancer, breast cancer, lymphoma, and leukemia.
Topoisomerase inhibitors interfere with enzymes called topoisomerases, which help separate the strands of DNA so they can be copied. They are used to treat certain leukemias, as well as lung, ovarian, gastrointestinal, and other cancers. Examples of topoisomerase I inhibitors include topotecan and irinotecan (CPT-11). Examples of topoisomerase II inhibitors include etoposide (VP-16) and teniposide. Treatment with topoisomerase II inhibitors increases the risk of a second cancer—acute myelogenous leukemia. Secondary leukemia can be seen as early as 2-3 years after the drug is given.
Mitotic inhibitors are often plant alkaloids and other compounds derived from natural products. They can stop mitosis or inhibit enzymes from making proteins needed for cell reproduction. These drugs work during the M phase of the cell cycle, but can damage cells in all phases. They are used to treat many different types of cancer including breast, lung, myelomas, lymphomas, and leukemias. These drugs are known for their potential to cause peripheral nerve damage, which can be a dose-limiting side effect. Examples of mitotic inhibitors include: the taxanes, such as paclitaxel (Taxol®) and docetaxel (Taxotere®); epothilones, which include ixabepilone (Ixempra®); the vinca alkaloids, such as vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®); and estramustine (Emcyt®).
Steroids are natural hormones and hormone-like drugs that are useful in treating some types of cancer (lymphoma, leukemias, and multiple myeloma), as well as other illnesses. When these drugs are used to kill cancer cells or slow their growth, they are considered chemotherapy drugs. Corticosteroids are commonly used as anti-emetics to help prevent nausea and vomiting caused by chemotherapy, too. They are also used before chemotherapy to help prevent severe allergic reactions (hypersensitivity reactions). Examples include prednisone, methylprednisolone (Solumedrol®) and dexamethasone (Decadron®).
Some chemotherapy drugs act in slightly different ways and do not fit well into any of the other categories. Examples include drugs such as L-asparaginase, which is an enzyme, and the proteosome inhibitor bortezomib (Velcade®).
Some other drugs and biological treatments are used to treat cancer, but are not usually considered “chemotherapy.” While chemotherapy drugs take advantage of the fact that cancer cells divide rapidly, these other drugs target different properties that set cancer cells apart from normal cells. They often have less serious side effects than those commonly caused by chemotherapy drugs because they are targeted to work mainly on cancer cells, not normal, healthy cells. Many are used along with chemotherapy.
As researchers have come to learn more about the inner workings of cancer cells, they have begun to create new drugs that attack cancer cells more specifically than traditional chemotherapy drugs can. Most attack cells with mutant versions of certain genes, or cells that express too many copies of a particular gene. These drugs can be used as part of primary treatment or after treatment to maintain remission or decrease the chance of recurrence. Only a handful of these drugs are available at this time. Examples include imatinib (Gleevec®), gefitinib (Iressa®), erlotinib (Tarceva®), sunitinib (Sutent®) and bortezomib (Velcade®).
Differentiating agents act on the cancer cells to make them mature into normal cells. Examples include the retinoids, tretinoin (ATRA or Atralin®) and bexarotene (Targretin®), as well as arsenic trioxide (Arsenox®).
Hormone therapy includes the use of sex hormones, or hormone-like drugs, that alter the action or production of female or male hormones. They are used to slow the growth of breast, prostate, and endometrial (uterine) cancers, which normally grow in response to natural hormones in the body. These cancer treatment hormones do not work in the same ways as standard chemotherapy drugs, but rather by preventing the cancer cell from using the hormone it needs to grow, or by preventing the body from making the hormones. Examples include: the anti-estrogens—fulvestrant (Faslodex®), tamoxifen, and toremifene (Fareston®); aromatase inhibitors—anastrozole (Arimidex®), exemestane (Aromasin®), and letrozole (Femara®); progestins megestrol acetate (Megace®); estrogens; anti-androgens—bicalutamide (Casodex®), flutamide (Eulexin®), and nilutamde (Nilandron®); and LHRH agonists—leuprolide (Lupron®) and goserelin (Zoladex®).
Some drugs are given to people with cancer to stimulate their natural immune systems to more effectively recognize and attack cancer cells. These drugs offer a unique method of treatment, and are often considered to be separate from chemotherapy. Compared to other forms of cancer treatment such as surgery, radiation therapy, or chemotherapy, immunotherapy is still relatively new. There are different types of immunotherapy. Active immunotherapies stimulate the body's own immune system to fight the disease. Passive immunotherapies do not rely on the body to attack the disease; instead, they use immune system components (such as antibodies) created outside of the body. Types of immunotherapies include: monoclonal antibody therapy (passive immunotherapies)—rituximab (Rituxan®) and alemtuzumab (Campath®); non-specific immunotherapies and adjuvants (other substances or cells that boost the immune response)—BCG, interleukin-2 (IL-2), and interferon-alpha; immunomodulating drugs—thalidomide and lenalidomide (Revlimid®); cancer vaccines (active specific immunotherapies)—although several vaccines are being studied, there are no FDA-approved vaccines to treat cancer (American Cancer Society, Inc. website, 2009).
The administration of botulinum toxin directly to cancer cells is also being used to treat the growth of tumors. The anaerobic, gram positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, referred to as botulinum toxin. To date seven immunologically distinct botulinum neurotoxins have been characterized: serotypes A, B, C1, D, E, F, and G. Of these, botulinum toxin serotype A is recognized as one of the most lethal naturally occurring agents.
It is thought that botulinum toxins bind with high affinity to cholinergic motor neurons, are transferred into the neuron and effectuate blockade of the presynaptic release of acetylcholine. All of the botulinum toxin serotypes are purported to inhibit release of acetylcholine at the neuromuscular junction. They do so by affecting different neurosecretory proteins and/or cleaving these proteins at different sites. For example, botulinum toxin serotype A is a zinc endopeptidase which can specifically hydrolyze a peptide linkage of the intracellular, vesicle associated protein SNAP-25. Botulinum toxin serotype E also cleaves the 25 kiloDalton (kD) synaptosomal associated protein (SNAP-25), however, serotype E binds to a different amino acid sequence within SNAP-25. It is believed that differences in the site of inhibition are responsible for the relative potency and/or duration of action of the various botulinum toxin serotypes.
Currently, botulinum toxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles. Botulinum toxin serotype A was approved in 1989 by the U.S. Food and Drug Administration (FDA) for the treatment of blepharospasm, strabismus, and hemifacial spasm in patients over the age of twelve. In 2000, the FDA approved commercial preparations of botulinum toxin serotype A and serotype B for the treatment of cervical dystonia, and in 2002 the FDA approved botulinum toxin serotype A for the cosmetic treatment of certain hyperkinetic (glabellar) facial wrinkles. In 2004, the FDA approved botulinum toxin for the treatment of hyperhidrosis. Non-FDA approved uses include treatment of hemifacial spasm, spasmodic torticollis, oromandibular dystonia, spasmodic dysphonia and other dystonias, tremor, myofascial pain, temporomandibular joint dysfunction, migraine, and spasticity.
Clinical effects of peripheral intramuscular botulinum toxin serotype A are usually seen within 24-48 hours of injection and sometimes within a few hours. When used to induce muscle paralysis, symptomatic relief from a single intramuscular injection of botulinum toxin serotype A can last approximately three months, however, under certain circumstances effects have been known to last for several years.
Despite the apparent difference in serotype binding, it is thought that the mechanism of botulinum activity is similar and involves at least three steps. First, the toxin binds to the presynaptic membrane of a target cell. Second, the toxin enters the plasma membrane of the effected cell wherein an endosome is formed. The toxin is then translocated through the endosomal membrane into the cytosol. Third, the botulinum toxin appears to reduce a SNAP disulfide bond resulting in disruption in zinc (Zn++) endopeptidase activity, which selectively cleaves proteins important for recognition and docking of neurotransmitter-containing vesicles with the cytoplasmic surface of the plasma membrane, and fusion of the vesicles with the plasma membrane. Botulinum toxin serotypes B, D, F, and G cause degradation of synaptobrevin (also called vesicle-associated membrane protein (VAMP)), a synaptosomal membrane protein. Most of the VAMP present at the cytosolic surface of the synaptic vesicle is removed as a result of any one of these cleavage events. Each toxin specifically cleaves a different bond.
The molecular weight of the botulinum toxin protein molecule, for all seven of the known botulinum toxin serotypes, is about 150 kD. Interestingly, the botulinum toxins are released by Clostridial bacterium as complexes comprising the 150 kD botulinum toxin protein molecule along with associated non-toxin proteins. Thus, the botulinum toxin serotype A complex can be produced by Clostridial bacterium as 900 kD, 500 kD and 300 kD forms. Botulinum toxin serotypes B and C1 are apparently produced as only a 500 kD complex. Botulinum toxin serotype D is produced as both 300 kD and 500 kD complexes. Finally, botulinum toxin serotypes E and F are produced as only approximately 300 kD complexes. The complexes (e.g molecular weight greater than about 150 kD) are believed to contain a non-toxin hemagglutinin protein and a non-toxin and non-toxic nonhemagglutinin protein. These two non-toxin proteins (which along with the botulinum toxin molecule can comprise the relevant neurotoxin complex) may act to provide stability against denaturation to the botulinum toxin molecule and protection against digestive acids when toxin is ingested. Additionally, it is possible that the larger (greater than about 150 kD molecular weight) botulinum toxin complexes may result in a slower rate of diffusion of the botulinum toxin away from a site of intramuscular injection of a botulinum toxin complex. The toxin complexes can be dissociated into toxin protein and hemagglutinin proteins by treating the complex with red blood cells at pH 7.3. The toxin protein has a marked instability upon removal of the hemagglutinin protein.
All the botulinum toxin serotypes are made by Clostridium botulinum bacteria as inactive single chain proteins which must be cleaved or nicked by proteases to become neuroactive. The bacterial strains that make botulinum toxin serotypes A and G possess endogenous proteases and serotypes A and G can therefore be recovered from bacterial cultures in predominantly their active form. By contrast, botulinum toxin serotypes C1, D, and E are synthesized by nonproteolytic strains and are therefore typically unactivated when recovered from culture. Botulinum toxin serotypes B and F are produced by both proteolytic and nonproteolytic strains and therefore can be recovered in either the active or inactive form. However, even the proteolytic strains that produce, for example, botulinum toxin serotype B only cleave a portion of the toxin produced. The exact proportion of nicked to unnicked molecules depends on the length of incubation and the temperature of the culture. Therefore, a certain percentage of any preparation of, for example, the botulinum toxin serotype B toxin is likely to be inactive, possibly accounting for a lower potency of botulinum toxin serotype B as compared to botulinum toxin serotype A. The presence of inactive botulinum toxin molecules in a clinical preparation will contribute to the overall protein load of the preparation, which has been linked to increased antigenicity, without contributing to its clinical efficacy.
In vitro studies have indicated that botulinum toxin inhibits potassium cation induced release of both acetylcholine and norepinephrine from primary cell cultures of brainstem tissue. Additionally, it has been reported that botulinum toxin inhibits the evoked release of both glycine and glutamate in primary cultures of spinal cord neurons and that in brain synaptosome preparations botulinum toxin inhibits the release of each of the neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP and glutamate.
High quality crystalline botulinum toxin serotype A can be produced from the Hall A strain of Clostridium botulinum with characteristics, of 3×107 U/mg, an A260/A278 of less than 0.60 and a distinct pattern of banding on gel electrophoresis. The known Shantz process can be used to obtain crystalline botulinum toxin serotype A, as set forth in Shantz, E. J., et al, Properties and use of Botulinum toxin and Other Microbial Neurotoxins in Medicine, Microbiol Rev. 56: 80-99 (1992). Generally, the botulinum toxin serotype A complex can be isolated and purified from an anaerobic fermentation by cultivating Clostridium botulinum serotype A in a suitable medium. Raw toxin can be harvested by precipitation with sulfuric acid and concentrated by ultramicrofiltration. Purification can be carried out by dissolving the acid precipitate in calcium chloride. The toxin can then be precipitated with cold ethanol. The precipitate can be dissolved in sodium phosphate buffer and centrifuged. Upon drying there can then be obtained approximately 900 kD crystalline botulinum toxin serotype A complex with a specific potency of 3×107 LD50 U/mg or greater. This known process can also be used, upon separation out of the non-toxin proteins, to obtain pure botulinum toxins, such as for example: purified botulinum toxin serotype A with an approximately 150 kD molecular weight with a specific potency of 1-2×108 LD50 U/mg or greater; purified botulinum toxin serotype B with an approximately 156 kD molecular weight with a specific potency of 1-2×108 LD5 U/mg or greater, and; purified botulinum toxin serotype F with an approximately 155 kD molecular weight with a specific potency of 1-2×107 LD5 U/mg or greater.
Already prepared and purified botulinum toxins and toxin complexes suitable for preparing pharmaceutical formulations can be obtained from List Biological Laboratories, Inc., Campbell, Calif.; the Centre for Applied Microbiology and Research, Porton Down, U.K.; Wako (Osaka, Japan), as well as from Sigma Chemicals of St Louis, Mo.
The pattern of toxin spread within a muscle has been demonstrated to be related to concentration, volume and location of injection site.
Several patents and applications relate to treating cancers with a neurotoxin and specifically a botulinum toxin. Uniformly, the methods directly deliver botulinum toxin to the cancerous cells with the goal of directly affecting the cancerous cells or their innervation. The goal has been to deliver the toxin into the cancerous cell to exert an effect, or to locally denervate a cancerous cell. By getting the toxin into a cell, botulinum toxin may inhibit the process of exocytosis from the cancer cell, which is the release of a cell's intracellular contents or vesicles into the extracellular space. These patents and applications pertain to the inhibition of exocytosis of a cancer cell and its reduced ability to divide and move. By locally denervating a cancer cell, it may become less active.
Patent application US 2005/0031648 A1, Methods for Treating Diverse Cancers, relates to the treatment of hyperplastic, precancerous or cancerous tissues with a botulinum neurotoxin by locally administering the botulinum toxin to the hyperplastic, precancerous or cancerous tissue or to the vicinity of cancerous tissue.
Patent application WO 2005/030248 relates to a method of increasing the entry of a Clostridium botulinum C3 exotransferase unit into cancer cells by linking the C3 to a cell-permeable fusion protein. The treatment pertains to the prevention of the cancer cell from contracting and spreading. The described compound specifically targets a cancer cell.
US 2002/0094339 A1, U.S. Pat. No. 6,565,870 B1 and U.S. Pat. No. 6,139,845 all relate to the treatment of tumors, cancers and disorders with a botulinum toxin. The toxin is injected directly into the diseased tissue to exert its effect on inhibiting exocytosis.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.